Chitin and chitosan are biopolymers, which typically are obtained from crustacean shell wastes, but can also be obtained from certain fungi. Chitosan may be prepared from chitin by chemical deacetylation. This is typically achieved by hydrolysing the N-acetyl linkage in chitin with concentrated alkali (40-50% NaOH or KOH). By definition, chitosan is generally described as a copolymer of D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc or NAG), which is insoluble in water at pH above 6.2—the isoelectric point of the free amine group—but dissolves at pH below 6.2 (See Scheme 1 and 2). In chitosan, 65-100% of the monomer units are D-glucosamine, which is usually described as 65-100% deacetylated chitin. Chemical and biological properties of chitosan are directly influenced by the degree of deacetylation (DDA) and degree of polymerisation (DP), i.e. the chain length of the polymer.
In solution at pH below 6.2, and when amine groups of the D-glucosamine residues are protonated, chitosan is a positively charged polymer. Being an amine, chitosan is a weak base and can form salts with acids, such as carboxylic and mineral acids. Most of these salts are water-soluble.
In its natural form, chitin is insoluble in water. However, it can be made water-soluble by partial deacetylation through alkali treatment [1]. Partially deacetylated chitin with DDA of 35-50% is soluble in water at a wide range of pH. This form of water-soluble chitin has been shown to be an excellent substrate for chitin converting enzymes [2, 3]. Moreover, preparation of water soluble chitin has shown to be a necessary step to retain high yield of chito-oligomers using chitinases, since insoluble chitin is hydrolyzed very slowly by chitinases [1].


Chito-oligomers (COs) and low molecular weight chitin and chitosan are shorter segments, made from the higher molecular weight polysaccharides by hydrolysis of the beta-(1,4)-bonds that link the monomers. Chito-oligomers refer herein to short to medium-length polymers, preferably having a degree of polymerization (DP) in the range of 2 to 50, corresponding to a molecular mass of about 360 to about 10.000 Da. COs made from water-soluble chitin (DDA 35-50%) maintain their water solubility. COs are made either chemically by using strong acids such as hydrochloric acid, catalysing the hydrolysis of the beta-(1,4)-bond at high temperatures, or by using enzymatic hydrolysis [4,5]. Enzymatic hydrolysis is favored since the process is easier to control and conditions are much milder and involve less risk of side reactions resulting in chemical modifications of the material.
Arthritis is a general term for inflammation of the joint(s), and sometime used to include all joint disorders. Osteoarthritis is the commonest form of joint disease in which there is damage to the surface of the joint and an abnormal reaction in the underlying bone. Other terms are used to describe this disease, such as ‘osteoarthrosis’, ‘arthrosis’ and ‘degenerative joint disease’. The disease mainly affects knees, hips, and hands (most common), as well as the foot and the neck and back. Rheumatoid arthritis is a common inflammatory disease of the joints, which causes inflammation of the lining membrane of the joint (synovium). This results in more swelling and other signs of inflammation than is usual in osteoarthritis, and can lead to severe damage of the joints.
Bioactivity of Chito-oligosaccarides:
Biological activity of chitin and chitosan is abundantly documented in the literature. Bioactivity studies have clearly demonstrated the importance of degree of polymerization (DP) as well as degree of deacetylation (DDA) [6]. In plants, the oligomers of DP 5-7 are more active than DP 1-4 [7]. The reason has been related to the ability of so-called chitinase-like proteins (CLPs) to bind chito-oligomers. These proteins share a high sequence homology and a structural relationship with family 18 chitinases [8]. The CLPs lack catalytic activity because of a single point mutation in their catalytic domain but they maintain their oligosaccharide binding ability, which usually involves 5-7 chito-oligosaccharide units.
N-Acetyl-Glucosamine, Chito-oligosaccharides and Hyaluronan: Glucosamine (GN or GlcN) is a modified glucose with NH2 replacing the OH group on the carbon two in the sugar molecule. In animal cells, glucosamine is only found in two forms; as glucosamine-6-phosphate (GN-6-P) and N-acetylglucosamine (NAG or GlcNAc). The amino sugar GN-6-P is synthesized from glutamine and fructose-6-phosphate (F-6-P). This reaction is catalysed by glucosamine synthase and is the rate limiting step in amino sugar biosynthesis. GN-6-P is the precursor to all hexosamines and hexosamine derivatives. GN-6-P can subsequently be acetylated by acetyl coenzyme A to N-acetyl glucosamine (NAG). NAG can subsequently be converted into N-acetyl galactosamine or N-acetyl mannosamine. These three amino sugars are important in glycosylation of proteins as well as building blocks for glycolipids, glycosaminoglycans (GAG), hyaluronan and proteoglycans. Hyaluronan (HA), the backbone of many proteoglycans, is a polysaccharide (up to 25,000 sugar units) composed of repeating disaccharide units of NAG and glucuronic acid (GlcA). HA is thought to be the earliest evolutionary form of GAG. HA is not only an important polysaccharide in cartilage, synovial fluid, viterous humor of the eye and in the skin of vertebrates, but may also play an important role in tissue organization, morphogenesis, cancer metastasis, wound healing and inflammation [9]. It is produced in large quantities during wound repair, and is an essential constituent of joint fluid (synovial fluid), where it serves as a lubricant [10]. NAG increases the synthesis of hyaluronan by mesothelial cells and fibroblasts in a dose-dependent manner [11]. HA is secreted from cells by an enzyme complex, named HA synthases (HAS) which is embedded in the plasma membrane [9]. These enzymes are thought to have evolved from chitin synthases or cellulose synthases [9]. A mouse HA synthase (HAS1) is capable to synthesize HA in vitro, when it is supplied with UDP-GlcA and UDP-NAG [12]. When HAS1 is incubated with UDP-NAG alone, it synthesizes chito-oligosaccarides (COs) [12]. A demonstration of similar activity of eukariotic HA synthases in vivo, would suggest novel functions for COs in mammals [9]. COs are produced in vivo during the development of vertebrates (Xenopus, zebrafish and mouse), where the chitinase-like DG42/HAS subfamily synthesizes both COs and HA during cell differentiation and the COs have been shown to be vital for a normal anterior/posterior axis formation in the late gastrla [9, 12-16], reviewed by [8].
Recent studies have suggested methods of treating arthritis by administration of glucosamine. These studies have shown that administration of glucosamine tends to normalize cartilage metabolism, inhibiting degradation, and stimulating the synthesis of proteoglycans, resulting in aortial restoration of the articular function. The therapeutic efficacy of treatment with glucosamine has been demonstrated in a number of animal and human studies.
U.S. Pat. No. 6,117,851 [17] teaches that (poly)-N-acetyl glucosamine (poly-NAG), i.e. chitin can be used to treat osteoarthritis and/or alleviate symptoms thereof. However, chitin, acting like insoluble fibre in the gut, is unlikely to be digested and absorbed. Also, due to its poor solubility in the gut environment, chitin is not likely to be efficiently hydrolysed by the recently discovered acidic mammalian chitinase (AMCase) [18] or intestinal bacteria producing lower molecular chitin fragments available for absorption. Partially deacetylated chitin however, is water-soluble at any pH and readily available as substrate for AMCase or intestinal flora.
Chito-oligomer Activity on Immune Response and Inflammatory Reactions —Chondrocytes and Macrophages:
Chitin and chitosan have been suggested to possess immunostimulating activity in mammals [19-22]. Also, chitin and chitosan have been studied in wound healing and artificial skin substitutes for some years [19-22]. In these studies, chitin and chitosan have showed a significant inhibitory effect on nitric oxide (NO) production by activated macrophages. Hexa-N-acetylchitohexaose (GlcNAc)6 and penta-N-acetylchitopentaose (GlcNAc)5 also inhibited NO production but with less potency. These results indicate that the positive effect of chitinous materials on wound healing is at least partly related to the inhibition of NO production by the activated macrophages [23]. It has also been shown that both glucosamine and N-acetylglucosamine inhibit NO production in normal human articular chondrocytes and that N-acetylglucosamine has a novel mechanism for the inhibition of inflammatory processes [24].
The chitinase-like protein YKL-40, also called human cartilage glycoprotein-39 (HC gp-39), is a member of family 18 chitinases [25]. YKL-40 is secreted by chondrocytes, synovial cells, and macrophages [26]. HC gp-39 (YKL-40) appears to be induced in aging human and young osteroarthritis patients [28]. It has been reported that YKL-40 has a role as an auto-antigen in rheumatoid arthritis (RA) [29-31] and it is expressed in diseased human osteoarthritic cartilage and osteophyte, but not in non-diseased tissue [32].